Purpose:
To determine the genetic cause of the disease in patients where clinical diagnosis was compatible with ABCA4-associated phenotypes (Stargardt disease, cone-rod dystrophy, bull’s eye maculopathy and atypical arRP), but where no two disease-associated ABCA4 mutations were identified.

Methods:
Detailed medical history and ophthalmic examination, including fundus photography, autofluorescence imaging, spectral-domain optical coherence tomography (SD-OCT) and, in some cases, ISCEV-standardized electroretinography, were performed on all patients. The ABCA4 gene was screened in all affected individuals with negative results. Subsequently, affected and available unaffected members from each family and sporadic cases were subjected to whole exome sequencing (WES) on Illumina platform. Possibly disease-associated variants were determined by filtering based on minor allele frequency and predicted pathogenicity. Variants were verified by Sanger sequencing followed by segregation analysis.

Results:
Ophthalmological examination identified 40 families and 26 sporadic cases with phenotypes compatible with STGD (42), CRD (6), arRP (3), and with BEM (15) all of whom harbored no or one disease-associated ABCA4 mutations after initial analysis. In total, 141 individuals were subjected to WES analysis. The definite causal gene was identified in 22/40 families. Analysis of 15/40 families resulted in several candidate genes, while no plausible candidate was found in 3 families. Of the 26 sporadic cases, 12 were solved, 9 had multiple plausible candidate genes, and 5 were impossible to solve. The causal genes for the solved cases belong to the following categories: 1) phenotypic expansion in known retinal disease genes (11 cases; CRB1, CRX, etc.); 2) revised clinical diagnosis or ambiguous phenotypes (12 cases; RS1, PRPH2, RP1L1, etc.); 3) new non-coding or re-assessed mutations in ABCA4 (7 cases); 4) new genes (RDH11, RAB28) in 3 families; 5) unusual clinical outcome, MMACHC, in one case.

Conclusions:
Whole-exome sequencing identified the causal gene in >50% of cases with phenotypes compatible with ABCA4-associated diseases. The genetic causes of ABCA4-like phenotypes range from new genes to unusual clinical manifestations of known genes.